The influence of a natural cross-linking agent (Myrica rubra) on the properties of extruded collagen fibres for tissue engineering applications
Post on 26-Jun-2016
k, Nf Ire
Article history:Received 26 April 2009
Keywords:Plant extractCollagen stabilisation
Extruded collagen bres have been shown to be a competitive biomaterial for both soft and hard tissuerepair. The natural cross-linking pathway of collagen does not occur in vitro and consequently reconstituted
cient strength. Numerous cross-linking approaches have been investigated
scaffold to achieve functional tissue reconstruction.
Materials Science and Engineering C 30 (2010) 190195
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j ourna l homepage: www.e lCollagen, under appropriate conditions of temperature, pH, ionicstrength, collagen concentration and composition and presence of otherconnective tissue macromolecules will spontaneously self-assemble toform microscopic brils, bril bundles and macroscopic bres thatexhibit D periodic banding patterns virtually indistinguishable fromnative bres when examined by electron microscopy . Theprinciple of self-assembly has been utilised to fabricate extrudedcollagen bres that closely imitate extracellular matrix assembliessuitable for both soft and hard tissue repair . Moreover, after therecent drawback of electro-spun collagen nano-bres [14,15], extrudedcollagen bres constitute the solely engineered brous scaffolds that
In every tissue engineering application, it is essential for the scaffoldto provide a mechanically stable construct upon which cells can attach,migrate and proliferate and therefore allow the formation of functionalneotissue. In vivo, the native cross-linking pathway of lysyl oxidaseimparts desired mechanical characteristics and proteolytic resistance onthe collagen bres in connective tissues . However, the lysyloxidase mediated cross-linking does not occur in vitro and consequentlycollagen constructs lack sufcient strength and may disintegrate uponhandling or collapse under the pressure from surrounding tissue uponimplantation. For this reason, a number of cross-linking approaches(chemical, physical and biological) have been investigated through theclosely emulate native tissues. Despite signica
This work was carried out at the School of ApplieNorthampton, UK. Corresponding author. Network of Excellence for Fun
Building, IDA Business Park, Newcastle Road, Dangan,Galway (NUIG), Galway, Ireland. Tel.: +353 0 9149 3166
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0928-4931/$ see front matter 2009 Elsevier B.V. Adoi:10.1016/j.msec.2009.09.017achieved, there are still many challenges in the engineering of this1. IntroductionMechanical propertiesThermal propertiesBiomaterialstabilisation effect of Myrica rubra on extruded collagen bres. Fibres treated with M. rubra exhibited higherdenaturation temperature (p
191D.I. Zeugolis et al. / Materials Science and Engineering C 30 (2010) 1901952. Materials and methods
All chemicals, unless otherwise stated, were purchased fromSigma-Aldrich, UK. The bovine Achilles tendons were kindly providedby the British Leather Research Centre (BLC; Northampton, UK).
2.2. Collagen preparation
A typical protocol for the extraction of collagen was employedas has been described in detail previously . Frozen bovineAchilles tendons were minced, washed in neutral phosphate buffersand suspended in 0.5 M ethanoic acid in the presence of pepsin(2500 U/mg, Roche Diagnostics, UK) for 72 h at 4 C. Consequently thecollagen suspension was centrifuged (12,000 g at 4 C for 45 min;Gr20.22 Jouan refrigerated centrifuge, Thermo Electron Corporation,Bath, UK) and puried by repeated salt precipitation (0.9 M NaCl),centrifugation and acid solubilisation (1 M ethanoic acid). Thenal atelocollagen collagen solution was dialysed (8000 Mw cut off)against 0.01 M ethanoic acid and kept refrigerated at 4 C untilused. The collagen purity was determined by SDS-PAGE analysis (90%type I) and its concentration was determined by hydroxyprolineassay (7 mg/ml).
2.3. Micro-bre fabrication and cross-linking
The procedure for bre formation has been described in detailpreviously  and was based on previous work [9,13,34]. Briey, a5 ml syringe (Terumo Medical Corporation UK Ltd, Merseyside, UK)containing the atelocollagen was loaded into a syringe pump system(KD-Scientic 200, KD-Scientic Inc., Massachusetts, USA) connectedto silicone tubing (Samco Silicone Products, Ltd., Warwickshire, UK) of30 cm in length and 1.5 mm in internal diameter. The pumpwas set toextrude at 0.4 ml/min. One end of the tube was connected to thesyringe pump with the other end placed at the bottom of a container.The collagen solution was extruded into a Fibre Formation Buffer(FFB) comprising of 118 mM phosphate buffer and 20% of polyeth-ylene glycol (PEG), Mw 8000 at pH 7.55 and 37 C. Fibres wereallowed to remain in this buffer for a maximum period of 10 min,followed by further incubation for additional 10 min in a FibreIncubation Buffer (FIB) comprising of 6.0 mM phosphate buffer and75 mM sodium chloride at pH 7.10 and 37 C. Thereafter, the breswere either incubated overnight at room temperature (RT) intodistilled water bath or cross-linked in aqueous 1% of formaldehyde or1% glutaraldehyde or 1% M. rubra (kindly provided by Prof. TonyCovington, University of Northampton, Northampton, UK). Finally, thebres were washed extensively in phosphate buffer saline (PBS), air-dried under the tension of their own weight and conditioned at RT at65% relative humidity for at least 48 h.
2.4. Denaturation temperature
The hydrothermal stability of the bres was determined using an822e Mettler-Toledo differential scanning calorimeter (Mettler-Toledo International Inc., Leicester, UK). Differential scanning calo-rimetry is a method widely used to study the thermal behaviour ofmaterials as they undergo physical and chemical changes uponheating. This method measures the heat ow necessary for heating ofthe sample with a constant temperature rate (C/min) . Drycollagen bres were hydrated overnight at RT in 0.01 M PBS at pH 7.4.The wet bres were removed and quickly blotted with lter paper toremove excess surface water and hermetically sealed in aluminiumpans. Heating was carried out at a constant temperature ramp (5 C/min) in the temperature range of 15 to 100 C, with an empty alu-
minium pan as the reference probe. The temperature of maximumpower absorption during denaturation (peak temperature) wasrecorded as the denaturation temperature [23,3840].
2.5. Mechanical testing and structural evaluation
Dry collagen bres were hydrated overnight at RT in 0.01 M PBS atpH 7.4. The wet bres were removed and quickly blotted with lterpaper to remove excess surface water. Stressstrain curves weredetermined in uniaxial tension using an Instron 1122 Universaltesting machine (Instron Ltd, Buckinghamshire, UK) operated at anextension rate of 10 mm/min. The gauge length was xed at 3 cm andsoft rubber was used to cover the inside area of the grips to avoiddamaging the bres at the contact points. Results obtained with bresthat broke at contact points with the grips were rejected. The cross-sectional area of each bre was calculated by measuring the diameterat four places along its longitudinal axis using a Nikon Eclipse E600optical microscope with a calibrated eyepiece (Nikon Instruments,Surrey, UK). It was assumed that the bres were circular for the cross-sectional area determinations. Surface and fractured ends of collagenbres that had been extended to failure were examined using aHitachi S3000N Variable Pressure Scanning Electron Microscope(Hitachi, Berkshire, UK). The following denitions were used tocalculate the mechanical data: stress at break was dened as the loadat failure divided by the original cross-sectional area (engineering-stress); strain at break was dened as the increase in bre lengthrequired to cause failure divided by the original length and moduluswas dened as the stress at 0.02 strain divided by 0.02.
2.6. Statistical analysis
Numerical data is expressed as meanSD. Analysis was per-formed using statistical software (MINITABTM version 13.1, Minitab,Inc.). One way analysis of variance (ANOVA) for multiple comparisonsand 2-sample t-test for pair wise comparisons were employed afterconrming the following assumptions: (a) the distribution fromwhich each of the samples was derived was normal (AndersonDarling normality test); and (b) the variances of the population of thesamples were equal to one another (Bartlett's and Levene's tests forhomogenicity of variance). Non-parametric statistics were utilisedwhen either or both of the above assumptions were violated andconsequently KruskalWallis test for multiple comparisons or MannWhitney test for 2-samples was carried out. Statistical signicancewas accepted at p
r ths (b
192 D.I. Zeugolis et al. / Materials Science and Engineering C 30 (2010) 190195denaturation temperature higher than the control (p
has also been shown to depend on the cross-linking method employed[27,42,71,72]. A compact inter-bre space was found for every treat-ment; indeed, it has been reported that very little available inter-brillarspace occurs in bres formed in vitro . The four fracture modesidentied are in accord with previous publications [9,27,70,7376] andtheir relative occurrence has been attributed to thehandling of thebreswhilst in the wet state, to the different degree of stretching of thedifferent layers of the bre, to the strain rate, or even to awswithin thebre structure.
4.2. Thermal analysis
polypeptides followed by the denaturation of the helical form .The collagengelatin transition is a melting process in which collagenchanges into a disorganised random coil [35,36,79]. Differentialscanning calorimetry is employed to evaluate the denaturationtemperature of fully hydrated biomaterials; scaffolds with meltingproles lower than 3940 C (body temperature) would indicatematerials unsuitable for biomedical applications since they woulddisintegrate upon implantation. Cross-linked and non-cross-linkedbres exhibited denaturation temperature higher than the bodytemperature making this scaffold an excellent choice for tissueengineering applications. Non-cross-linked bres had a denaturationtemperature higher than any other non-cross-linked collagenouspreparation (e.g. gels, lms, sponges) due to the increased energy ofcrystallisation derived from the interaction between the closelypacked molecules in the bre form [58,78,8082]. The presence ofPEG could also be responsible for the increased denaturationtemperature of the bres compared with that of collagen sponges orlms, where no added polymer was present. Indeed, polymers mayincrease the denaturation temperature by shifting the equilibriumbetween native and denaturated forms of collagen towards a morecompact native form by steric exclusions .
The different cross-linking methods employed in this studybrought about different thermal stabilities. It has been reported thatsamples with higher denaturation temperature have more hydrogenbonds and/or fewer hydrophobic bonds than samples with lowerdenaturation temperatures [86,87]. Moreover, it has been shown thatthe denaturation temperature depends on the size of the co-operatingunits, the larger the unit, the slower the kinetics and the higher theshrinkage temperature [76,88]. Indeed, the complex structure of M.
Fig. 2. Typical j-shape stressstrain curves of rehydrated extruded collagen bres wereobserved. The curves exhibited a diameter dependent variation; thin bres exhibited ahigh stress/low strain graph (I), whilst thick bres demonstrated a low stress/highstrain graph (II).
193D.I. Zeugolis et al. / Materials Science and Engineering C 30 (2010) 190195When collagen is heated in a hydrated state, the crystalline rigidtriple helical molecule denaturates over a narrow range of tempera-tures, the mid-point of which is referred to as denaturationtemperature (TD) and results in the destruction of its tertiary structureand biological function . The denaturation of the triple helix hasbeen shown to be a two-stage process starting with separation of theFig. 3. Fitting a linear regressionmodel between stress at break andwet bre diameter, strongthe stress at break was apparent for every treatment.rubra brought about the highest denaturation temperature, whilstbetween the aldehydes, glutaraldehyde conveyed higher denatur-ation temperature. In fact, it has been shown that the nature of thebonds formed and the stability of the cross-links introduced vary withthe aldehyde used, which has been attributed to the structuralchanges associated with the collagenaldehyde reaction . More-over, inter-brillar cross-links have not been described for
correlations were obtained and an inverse relationship between the bre diameter and
thick bres exhibit longer toe regions than their thinner counterpartswithin the same treatment.
194 D.I. Zeugolis et al. / Materials Science and Engineering C 30 (2010) 190195The stress, force and modulus values were increased after cross-linking for all treatments. We propose that residual water moleculeswithin the brous structure could be responsible for the observedincreased in the aforementioned values. Indeed, in non-cross-linkedbres water molecules could break down the hydrogen and theelectrostatic bonds that hold collagen brils together  and theinter- and intra-molecular hydrogen bonds and that control HOHcollagen bonds, i.e. the number and the length of distance betweenthe protein chains [72,117]. It is likely that after cross-linking, theseformaldehyde and it does not co-polymerise as demonstrated withglutaraldehyde and subsequently cannot bind side-by-side brils .Furthermore, formaldehyde does not introduce bulky polymericadducts into the bril structure, as has been shown for glutaraldehyde. Cross-linking increased the denaturation temperature of thebres due to the better packing and stabilisation of the helices andconsequently decreased the enthalpy of denaturation in all cases[36,58,78,8082]. However, this transition is a bulk response and doesnot reect the exact number or location of the cross-links [38,91].
4.3. Biomechanical evaluation
Tensile testing was performed to analyse the biomechanicalproperties of the collagen matrices. In vivo, the primary mechanicalstrength of individual collagen molecules depends upon the extra-cellular formation of triple helical molecules that self-assemble intocollagen brils and are stabilised by intra- and inter-molecular cross-links between the adjacent helical molecules . The collagennetwork is primarily responsible for the mechanical properties ofcollagenous tissues, especially for tissues that are exposed to repeatedtensile forces [20,50,51,93,94]. Uniaxial tensile tests of rehydratedcollagen bres produced j-shape stressstrain curves that have beenreported for native tissues [95,96] and extruded collagen bres[27,48,70,97]. Similar curves have also been reported for semi-crystalline polymers that yield and undergo plastic ow . Theyielding mechanism involves some form of ow that occurs withinthe bre, possibly inter-brillar slippage, which plays an importantrole in the tensile deformation of aligned connective tissues such astendons . In all cases, the slope of the stressstrain curve increaseswith strain, a characteristic of native and in vitro producedcollagenous structures . The low modulus of the toe regionthat gives rise to a non-linear stressstrain curve in native tissues hasbeen attributed to the reorientation/alignment and un-crimping ofthe collagen brils, as well as the initiation of stretching of the triplehelix, the non-helical ends and the cross-links .
Strong inverse correlations between bre diameter and stress atbreak were obtained suggesting that by controlling the bre diameter,extruded collagen bres can be produced with variable strength tomatch the tissue to be replaced. Three different mechanisms couldbe responsible for the observed correlations: (a) the tensile strengthincreases as the cross-sectional area decreases because there is lesschance for defects in thinner sections , (b) as the brediameter decreases, improved longitudinal alignment takes placethat enhances strong interactions between the collagen brils[96,97,110,111]. These strong interactions are manifested in thestressstrain curves with the short toe region observed for the thinbres, whilst the looser interactionswould tend to give rise to a longertoe region for the thick bres, (c) since water acts as a plasticiser forbiopolymers  and as a mild plasticiser for collagen , watermolecules are not removed during air-drying  and inexibility ofbres is achieved by removal of water from the brous structure ,it is realistic to presume that thick bres could have increased watercontent, whilst thin bres have absorbed less water and that is whywater-binding sites are unavailable to bond inter-molecularly andtherefore they stiffen the collagen triple helix and prevent slippageand translation to occur between neighbouring molecules .
Table 1 clearly demonstrates that different cross-linking approaches,due to the different chemistry that is involved in the stabilisationprocess, lead to diverse mechanical properties. In fact, it is long knownthat even between aldehydes, their ability to cross-link collagen withrespect to thenumberof cross-links introduced and their stabilitydiffersconsiderably among them . Formaldehyde treatment does notresult in inter-brillar cross-links; does not co-polymerise as has beendemonstrated with glutaraldehyde; and does not introduce bulkypolymeric adducts into the bril structure as has been shown forglutaraldehyde [89,90]. Glutaraldehyde on the other hand is abiofunctional cross-linking agent that can self-polymerise and conse-quently create a three-dimensional network with long-range cross-links spanning larger gaps, which can affect the collagen bresproperties by stiffening and strengthening the bres [47,48]. M. rubratannin molecule has highly nucleophilic reaction activity and it can becovalently bonded to amino groups of collagen molecules by reactionwith aldehyde [119,120].
The most signicant nding of this study is that extruded collagenbres were produced with mechanical and structural propertiesclosely matching native tissues. For example, human anterior cruciateligament, rat tail tendon, bovine and rabbit Achilles tendons havebeen shown to have diameter ranging from 20 to 400 m, that canwithstand mechanical loads from 15 to 53 MPa and exhibit strain atbreak from 7 to 40% [29,48,75,97,110,111,121,122]. Fixation using M.rubra yielded bres with properties falling within this range. It hasbeen reported that plant extracts exhibit lower cytotoxicity thanglutaraldehyde and remarkable resistance to degradation and as suchas promising strategies for functional tissue engineering applications[30,5557,62,123].
This work investigated the inuence of M. rubra on the propertiesof extruded collagen bres. Results reported demonstrate that theproduced bres are characterised by thermal, structural, physical andmechanical properties that arematching native tissues such as tendonand anterior cruciate ligament. Therefore, stabilisation usingM. rubracould be a valuable alternative to aldehyde approaches for the con-struction of three-dimensional scaffolds that would imitate nativeextracellular matrix assemblies.
The authors would like to thank Mrs. P. Potter, Ms. S. Lee, Mrs.T. Hayes and Mr. L. Stathopoulos for excellent technical assistance;Dr. S. Jeyapalina and Dr. P. Antunes for their useful discussion; andProf Tony Covington for sample donation. Dimitrios Zeugolis isgrateful to The University of Northampton and EPSRC for nancialsupport.
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The influence of a natural cross-linking agent (Myrica rubra) on the properties of extruded col.....IntroductionMaterials and methodsMaterialsCollagen preparationMicro-fibre fabrication and cross-linkingDenaturation temperatureMechanical testing and structural evaluationStatistical analysis
ResultsMatrix morphologyThermal propertiesBiomechanical analysis
DiscussionMatrix morphologyThermal analysisBiomechanical evaluation